Phosphorylation of ribosomal protein S6 is elevated in polyomavirus-infected cells. This elevation results only in part from activation of S6 kinase activity. These effects appear to reflect independent activities of wild-type middle T antigen. Hr-t mutant NG59, encoding a defective middle T protein, and mutant Py8O8A, encoding no middle T protein, were unable to induce S6 kinase activity or elevate S6 phosphorylation. Two other site-directed mutants encoding altered middle T proteins did elevate S6 phosphorylation while only weakly stimulating S6 kinase activity. These results suggest at least two independent pathways leading to elevation of S6 phosphorylation. One pathway leads to induction of S6 kinase activity following activation of pp60`cs by transformation-competent middle T antigen. Another pathway operates independently of S6 kinase induction and can be regulated by transformation-defective middle T mutants such as Py1387T. This mutant, encoding a truncated middle T protein that failed to associate with the plasma membrane and to activate pp60C"sc, caused increased levels of S6 phosphorylation without detectably increasing S6 kinase activity. The ability of mutants such as Py1387T to induce S6 phosphorylation correlated with their ability to increase phosphorylation of VP1, an event linked to maturation of infectious virions.Transformation of cells by polyomavirus requires the interaction of the polyomavirus middle T antigen with the cellular proto-oncogene product pp6Oc-src (15,16). This interaction leads to the activation of the intrinsic tyrosine kinase activity of pp60c-src and ultimately to transformation (10,14). Although pp60c-src activation is obligatory for transformation, mutant middle T's exist which interact with and activate pp6Oc-src yet fail to transform infected cells. Full transformation in culture and tumor induction in mice may require the presence of an additional activity in middle T-pp60csrc immune complexes which phosphorylates phosphatidylinositol (PI) (27, 51; D. Talmage, R. Freund, A. Young, C. Dawe, and T. Benjamin, manuscript in preparation).Polyomavirus hr-t mutants encoding defective middle T and small T antigens grow poorly on NIH 3T3 cells, yielding bursts which are only 2 to 5% the size of those of wild-type virus (22). The low burst size results from inefficient or unstable assembly of viral components into infectious virions and is associated with underphosphorylation of the major viral capsid protein, VP1 (19,20). Thus, the role of the hr-t gene in viral growth lies in inducing cellular "permissivity factors" which act, at least in part, to bring about stable phosphorylation of VP1. Activation of this phosphorylation pathway in NIH 3T3 cells does not require detectable interaction of middle T with pp60c-src and therefore represents a middle T function which is separate from its known function during transformation (R. Garcea, D. Talmage, R. Freund, A. Harmatz, and T. Benjamin, manuscript in preparation).The ribosomal protein S6 is highly phosphorylated in growing cell...